PROJECT SUMMARY The prevalence of Neisseria gonorrhoeae (Ng), its associated morbidity, and the emergence of untreatable strains, support a critical need for new preventative and therapeutic strategies for this important human- pathogen. Such developments require a complete understanding of the pathobiology of this archetypal, host- adapted pathogen. We show that phase-variable, glycan post-translational modifications on Ng surface appendages, pili (fimbriae), play a vital role in human infection. Complement receptor 3 (CR3) is an important pattern recognition receptor that is the key receptor mediating Ng colonization of human cervical mucosa. CR3 contains an ?I-domain? region, which is known as a binding site for protein ligands. However, we found that Ng binding to the I-domain is mediated by the pilin-linked glycan (PLG). This is a seminal finding in innate immunity, as all previous literature ascribe CR3 carbohydrate binding to a separate, so called lectin domain. This was also the first demonstration that post-translational modifications made to a bacterial protein modulate pathogenesis. These findings will impact our understanding of microbial pathogenesis and innate immune responses. The goals of the present application are to define the specific contribution of the six, naturally occurring, PLG structures in mediating the Ng-CR3 I-domain interaction and to determine the biological relevance of each of these interactions to infection in females. Guided by strong preliminary data, we hypothesize that variable PLG structures initiate key, but highly divergent, outcomes with CR3 I-domain engagement. We will resolve our hypothesis through two specific aims: Aim 1) Define the effect of variation in pilin glycan structure on direct PLG-I-domain interactions; we will define the specific molecular interactions occurring with the CR3 I-domain for the six natural Ng PLG structures. Aim 2) Define the effect of PLG-I- domain interactions on Ng pathogenesis and CR3 function; we will define targeted epithelial cell responses to CR3 I-domain engagement by Ng that bear different PLG structures and their effect on Ng pathogenesis. Our approach is innovative in using biologically relevant human primary cervical cells, both alone and in co-culture with phagocytes, combined with low passage Ng isolates. This ensures that data obtained are relevant to human processes. By defining the effector functions controlled by I-domain lectin activity on primary human epithelial cells, we will provide critical new information regarding Ng pathogenesis, CR3 function, and the fundamental mechanisms that govern human cervical mucosal immunity. Moreover, we will define the utility of targeting the PLG-CR3 I-domain interaction as a new, and improved, host-targeted approach to treat and/or prevent Ng disease in women. Our studies will very likely impact work on other human pathogens and on innate immunity, broadening the significance of our outcomes. We have extensive experience in defining the molecular mechanisms of Ng pathogenesis. We are ideally placed to do this work in having discovered CR3 as the key receptor for Ng cervical infection and the biosynthetic pathways for pili glycosylation.